(Re-)Directing Oligomerization of a Single Building Block into Two Specific Dynamic Covalent Foldamers through pH

Dynamic foldamers are synthetic folded molecules which can change their conformation in response to an external stimulus and are currently at the forefront of foldamer chemistry. However, constitutionally dynamic foldamers, which can change not only their conformation but also their molecular constitution in response to their environment, are without precedent. We now report a size- and shape-switching small dynamic covalent foldamer network which responds to changes in pH. Specifically, acidic conditions direct the oligomerization of a dipeptide-based building block into a 16-subunit macrocycle with well-defined conformation and with high selectivity. At higher pH the same building block yields another cyclic foldamer with a smaller ring size (9mer). The two foldamers readily and repeatedly interconvert upon adjustment of the pH of the solution. We have previously shown that addition of a template can direct oligomerization of the same building block to yet other rings sizes (including a 12mer and a 13mer, accompanied by a minor amount of 14mer). This brings the total number of discrete foldamers that can be accessed from a single building block to five. For a single building block system to exhibit such highly diverse structure space is unique and sets this system of foldamers apart from proteins. Furthermore, the emergence of constitutional dynamicity opens up new avenues to foldamers with adaptive behavior.

3,5-Bis(tritylthio)benzoic acid was synthesized via a previously reported procedure. 1  trifluoroacetic acid (TFA) were obtained from Sigma-Aldrich. Ultrapure water from a Milli Q water purification system (Burlington, USA) was used throughout. Other materials used for synthesis were commercially available and used as received. NMR spectra were recorded on Bruker Avance 600 MHz spectrometers. HRMS spectra were recorded on an Orbitrap Fusion Lumos Tribrid Mass Spectrometer.

Peptide synthesis
The synthesis of building blocks were performed using Fmoc solid-phase peptide synthesis on Wang resin (for building block 1, 2, and 3) or Rink Amide AM resin (for building block 4).
Specifically, for the synthesis of L-peptide acids, pre-loaded Fmoc-amino acid-Wang resins were S4 used. For the synthesis of D-1, Wang resin was brominated and loaded with Fmoc-D-Lys(Boc)-OH according to a standard protocol. 2 Fmoc deprotection steps were carried out with 20% piperidine in DMF (2×5 min). In the coupling of subsequent amino acids or 3,5bis(tritylthio)benzoic acid, 3-fold Fmoc-protected amino acids or 3,5-bis(tritylthio)benzoic acid in the presence of 3-fold HBTU, 3-fold HOBT and 8-fold NMM were used. Deprotection from the resin and removal of the protecting groups was performed using a cocktail of 94% TFA, 2.5% EDT, 2.5% water and 1% TIS for 2 h. The solution was then filtered, the filtrate was vacuum-dried Collector III) equipped with a NUCLEODUR C18 HTec column (Macherey-Nagel, 21×125 mm, 5 µm). The building blocks and cyclic compounds were obtained at a purity higher than 95%. Salt exchange from TFA to HCl was performed twice by treating purified macrocycles with excess 0.1 M HCl and subsequent freeze-drying.

Library preparation
Building blocks (final concentration 1.0 mM) were dissolved in phosphate buffer (25 mM or 50 mM). All the libraries were set up in an HPLC vial (12 × 32 mm) with a Teflon-coated screw cap.
All the HPLC vials were equipped with a cylindrical stirrer bar (2 × 5 mm, Teflon coated) and stirred at 150 r.p.m. using an IKA RCT basic hot plate stirrer. All experiments were performed at 40 °C.

UHPLC analysis
UHPLC analyses were performed on a Shimadzu LC-40D XR UHPLC system. The separation systems were all equipped with a photodiode array detector set at a detection wavelength of 254 nm. Samples were analyzed on a HALO peptide ES-C18 column (160 Å, 2 µm, 2.1 × 150 mm), using water (eluent A) and acetonitrile (eluent B), which each contained 0.1% v/v trifluoroacetic acid as the modifier. A flow rate of 0.2 mL min -1 and a column oven temperature of 30 °C were applied. Gradient: 0-1-12-12.2-16 min, 5%-15%-60%-5%-5%B. Sample preparation was performed by diluting 5.0 µL of the library with 30 µL of doubly distilled water. HPLC injection volume is 5 µL.

UPLC-MS analysis (for DCLs made from building block 2 and 3) were carried out on a Waters
Acquity UPLC H-class system coupled to a Waters Xevo-G2 TOF mass spectrometer. DCLs made from building block 4 were analyzed on an UltiMate 3000 UHPLC system equipped with a diodearray detector and connected to an LCQ Fleet mass spectrometer.

CD spectroscopy
Spectra were recorded on a Jasco J-810 spectrometer with a Peltier temperature controller. Heatcool cycles were applied from 20 to 90 °C in steps of 10 °C at a rate of 1 °C min -1 and maintained for 2 min at every temperature before measuring. Spectra were obtained as averages of three measurements from 200 to 400 nm with a scanning speed of 150 nm min -1 and a bandwidth of 1 nm. A quartz cuvette with a 1 cm path length was used for the measurements. The purified 19 and 116 were redissolved in 25 mM phosphate buffer at pH 8.2 and pH 6.0, respectively, for the temperature dependent CD measurements. The concentration of all samples was kept as 0.10 mM in building block.

pH switching cycles
The buffer exchange was performed using a 3K centrifugal filter (Amicon Ultra-0.5 mL). Briefly, the library solution was added onto the centrifugal filter which was centrifuged at 10,000 r/min for 10 min. The concentrated sample was washed twice with water and then diluted with phosphate buffer of a different pH. After one day, the library was analyzed by UHPLC.

pH titration
The purified samples were dissolved in water (final concentration 0.51 mM in building block, 1.5 mL) and added into a 2 mL polypropylene plastic tube containing a Teflon-coated magnetic stirring bar (5×2 mm). For the titration of monomer, tetramer and 9mer, 0.5 M HCl was first added to the solution step by step to lower the pH to ~2.7. The titration of the 16mer was started with a freshly prepared solution of 16mer in water without reducing the pH. Small aliquots (2 or 5 µL) of 0.02, 0.1 or 0.5 M NaOH solution were then added to the samples and the pH was monitored using a Mettler Toledo SevenCompact pH meter with an InLab® Ultra-Micro-ISM sensor. Every pH point was measured twice to confirm that the reading had stabilized. Every sample was titrated at least in duplicate.

Crystallization of 19 and 116
Aqueous solutions of L-19 and D-19 were prepared as HCl salt and dissolved using pure water to a final concentration of 25 mg/mL. Aqueous solutions of L-116 were prepared by dissolving the lyophilized powder using pure water and 3 µL of 1 M HCl to a final concentration of 25 mg/mL.

Data collection and structure determination of L/D-19
The X-ray diffraction data was collected at the ID23-1 beamline 3

in the European Synchrotron
Radiation Facility (ESRF), Grenoble. Diffraction data was measured at T = 100 K, dmin = 1.15Å, λ = 0.6888Å. The crystal was exposed for 0.01 s and 0.2° oscillation per frame and a rotation pass of 360° was measured using a Dectris Pilatus 6M detector. Diffraction data was processed using the autoPROC pipeline. [4][5][6][7][8] The crystal belonged to the Triclinic space group P1 with unit cell (2), γ = 82.314° (2); V = 15621 (7) Å 3 and 2 molecules per asymmetric unit (Z = Z' = 2). The S10 structure was solved with the program SHELX 9 and refined by full-matrix least-squares method on F 2 with SHELXL-2014 10 within Olex2 11 ( Figure S28). After each refinement step, visual inspection of the model and the electron-density maps were carried out using Olex2 11 and Coot. 12 The initial structure revealed most of the main-chain atoms of an L-19 macrocycle. After several iterations of least-squares refinement the main-chain trace improved for a second macrocycle L-19. All side chains of phenyl carboxylate and lysine were observed to be disordered and were either omitted or refined with partial occupancy and isotropic displacement parameters. AFIX, DFIX and FLAT instructions were used to improve the geometry of molecules. Constraints and restraints on anisotropic displacement parameters were implemented with EADP, DELU, SIMU, RIGU and ISOR instructions. After several attempts to model the disordered side chains, the SQUEEZE 13 procedure was used to flatten the electron density map. Very disordered side chains and solvent molecules were removed. Hydrogen atoms were not added due to the poor quality of the data.

Data collection and structure determination of L-116
The X-ray diffraction data was collected at the P13 beamline 14 operated by EMBL Hamburg, at the PETRA III storage ring (DESY, Hamburg) with a Dectris Pilatus 6M detector. Diffraction data were measured at T = 100 K, dmin = 1.15 Å, λ = 0.82656 Å. The crystal was exposed for 0.04 s and 0.15° oscillation per frame. 2800 images were collected in a sweep with a total exposure time of 112 s. Diffraction data was processed using the program CrysAlis Pro . 15 The crystal belonged to the Triclinic space group P1 with unit cell parameters: a = 25.676 (7)  and refined by full-matrix least-squares method on F 2 with SHELXL-2014 10 within Olex2 11 ( Figure   S29). The initial structure revealed all main-chain atoms and several side chains of two L-116 S11 macrocycles. After each refinement step, visual inspection of the model and the electron-density maps were carried out using Olex2 11 and Coot. 12 Some side chains of phenyl carboxylate and lysine were observed to be disordered and were either omitted or refined with partial occupancy and isotropic displacement parameters. AFIX, DFIX and FLAT instructions were used to improve the geometry of molecules. Constraints and restraints on anisotropic displacement parameters were implemented with EADP, DELU, SIMU, RIGU and ISOR instructions. After several attempts to model the disordered side chains, the SQUEEZE 13 procedure was used to flatten the electron density map. Very disordered side chains and solvent molecules were removed. Hydrogen atoms were placed at idealized positions except for those at disordered/missing side chains.
Statistics of data collection and refinement are described in Table S1. The final cif files were checked using IUCr's checkCIF algorithm. Due to large volume fractions of disordered solvent molecules, weak diffraction intensity and poor resolution, a number of A-and B-level remain in the checkCIF file. These alerts are inherent to the data and refinement procedures. They are listed below and have been divided into two groups. The first group illustrates weak quality of the data and refinement statistics if compared to that expected for small molecule structures from highly diffracting crystals. The second group is connected to decisions made during refinement and explained below. Atomic coordinates and structure factors for L/D-19 and L-116 were deposited in the Cambridge Crystallographic Data Centre (CCDC) with accession codes 2183369 and 2183384 respectively. The data is available free of charge upon request (www.ccdc.cam.ac.uk/).

CheckCIF validation of L/D-19:
Group 1 (these alerts illustrate weak quality of the data and refinement statistics if compared to that expected for small molecule structures from highly diffracting crystals): THETM01_ALERT_3_A The value of sine(theta_max)/wavelength is less than 0.550 S12 This positive residual density corresponds to an S atom (S18A) that was anisotropically refined. The peak remained despite attempts to improve geometry and temperature parameters.

CheckCIF validation of L-116:
Group 1 (these alerts illustrate weak quality of the data and refinement statistics if compared to that expected for small molecule structures from highly diffracting crystals): THETM01_ALERT_3_A The value of sine(theta_max)/wavelength is less than 0. These belong to the disordered peptide sidechains that were refined with isotropic displacement parameters. S14  Figure S1. 1